Accepted Article
Received Date : 25-Mar-2014 Revised Date : 26-May-2014 Accepted Date : 30-May-2014 Article type
: Original Articles
Hepatitis E infection in patients with severe acute alcoholic hepatitis HAIM-BOUKOBZA Stéphanie (1,2,3,6), COILLY Audrey (2,3,4,6), SEBAGH Mylène (3,5,6), BOUAMOUD Mouna (4), ANTONINI Teresa (3,4,6), ROCHE Bruno (3,4,6), YORDANOVA Olga (1), SAVARY Janine (1), SALIBA Faouzi (2,3,4,6), DUCLOS-VALLEE Jean-Charles (2,3,4,6), SAMUEL Didier (2,3,4,6), ICHAI Philippe (2,3,4,6), ROQUE-AFONSO Anne-Marie (1,2,3,6)
1
AP-HP, Hôpital Paul Brousse, Virologie, CNR des hépatites A et E, Villejuif, 94804 France
2
Univ Paris-Sud, UMR-S 785, Le Kremlin Bicêtre, 94275 France
3
INSERM U785, Villejuif, 94804 France
4
AP-HP, Hôpital Paul Brousse, Centre Hépato-Biliaire, Villejuif, 94804 France
5
AP-HP, Hôpital Paul Brousse, Service d’anatomie pathologie, Villejuif 94804
6
Hepatinov, Villejuif F94800, France
Corresponding author: Mailing address : Pr ROQUE-AFONSO Anne-Marie 12-14 av Paul Vaillant Couturier 94804 Villejuif Cedex France This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/liv.12610 This article is protected by copyright. All rights reserved.
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[email protected] Phone : + 33 1 45 59 37 21 Fax : + 33 1 45 59 37 24
ABSTRACT: BACKGROUND: Hepatitis E virus (HEV) infection is a known cause of acute on chronic liver failure in developing countries, but its implication in Western countries remains unknown.
AIMS: To assess HEV burden in the setting of severe acute alcoholic hepatitis (AAH).
METHODS: Patients admitted for severe AAH from 2007 to 2013, with available sera and histologically proven AAH, were included and managed according to current European guidelines. At admission, clinical and biological characteristics were collected; HEV serology and RNA detection were retrospectively performed.
RESULTS: Eighty-four patients were included. Mean age was 50.8±9.6 years, 65.5% were male, 91.7% were cirrhotic and 33.3% presented with encephalopathy. Mean MELD and Maddrey scores were respectively 32.4±11.4 and 73.3±37. Liver biopsy showed mild, moderate and severe hepatitis in 25 (29.8%), 23 (27.4%) and 32 (38.1%) patients, respectively. Steroids were given to 61 patients (72.6%) of whom 35 (57.4%) presented corticoresistance (mean Lille score: 0.78±0.21). During hospitalization, 24 patients (28.6%) died and 11 (13.1%) were transplanted. Three patients (3.6%) presented markers of acute HEV infection and 21 (25%), markers of past HEV infection. Patient with acute infection were men, cirrhotic, and 2/3 presented with encephalopathy. Steroids were given to 2 patients without any response. The third patient died. None were transplanted.
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CONCLUSIONS: A substantial proportion of patients with severe AAH had markers of acute HEV infection, with similar clinical presentation and outcomes. Larger studies are needed to evaluate HEV impact on AAH management, resistance to steroids, and outcome.
Key words: hepatitis E, acute alcoholic hepatitis, Lille score, corticoresistance, Maddrey score, MELD score, cirrhosis.
ARTICLE Alcoholic liver disease is the most prevalent cause of advanced liver disease in Europe (1). Hepatic manifestations include alcoholic fatty liver disease, acute alcoholic hepatitis (AAH) and cirrhosis. The majority of patients with AAH have a history of heavy alcohol use and concomitant cirrhosis is present in about 50%-60% cases (2). The clinical syndrome is characterized by a recent onset of deep jaundice, which can lead to an end stage liver failure. Symptoms are usually non-specific including fatigue, fever, ascites and encephalopathy. The diagnosis is based on histological features (i.e. steatosis, hepatocyte injury and neutrophilic infiltration). The overall 30-day mortality rate is approximately 15%. However, in patients with severe liver disease defined by a Maddrey’s discriminant (MDF) function ≥32, the rate can reach 50 % (3, 4). Short-term prognosis of patients with a severe AAH has been improved with steroid use (5).
Hepatitis E virus (HEV) infection is a cause of acute liver injury. In low-income countries with poor sanitation and hygiene, HEV is a common cause of acute hepatitis transmitted through contaminated water. In these countries, infection is due to genotypes 1 and 2 that exclusively infect humans. In industrialized countries, HEV is responsible for sporadic cases This article is protected by copyright. All rights reserved.
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due to genotypes 3 and 4 that also infect other animals, mainly pigs, and the transmission appears to be zoonotic or foodborne (6). HEV infection has been shown to account significantly for acute-on-chronic liver failure (ACLF), predominantly in patients from the Indian subcontinent, and it was shown that cirrhotic patients who experienced a superimposed HEV infection had a higher risk of rapid decompensation and death (7). The occurrence and outcome of HEV infection in patients presenting with ACLF is still poorly described in Western countries, especially in the population of patients with AAH (8, 9). Nevertheless, several facts argue for a systematic screening of HEV infection in patients with decompensated alcoholic liver disease in Western countries: 1-as in endemic countries, HEV infection could enhance acute or sub-acute liver failure in patients with pre-existing liver disease (9); 2-mortality due to chronic liver disease has been linked to the consumption of pork and alcohol in developed countries (10, 11); 3-there is a potential risk of developing a chronic HEV infection considering the immunosuppression status of AAH patients due to steroids (12). Hepatitis E is still not commonly diagnosed in the first line of ACLF in most centers, and is often an exclusion diagnosis (13, 14). The aim of this retrospective study was to assess the frequency of HEV infection and its prognosis in patients admitted for severe AAH in a single center.
PATIENTS AND METHODS: Patients’ selection All patients admitted in the hepato-biliary center of the Paul Brousse hospital from 2007 to 2013 for severe AAH were considered for the present study. Severity was defined as a MDF
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≥ 32 and/or a MELD score ≥ 20. Patients previously transplanted were excluded and only patients with histologically proven AAH and available serum were included.
Clinical management of AAH AAH management was based on current guidelines (15, 16). Psychotherapy to maintain abstinence was early initiated. A transjugular liver biopsy was systematically considered when MDF was ≥ 32. Patients with severe clinical presentation (MDF ≥ 32) were considered for treatment with prednisolone 40mg/day during 28 days in case of histologically proven AAH, whatever the degree of histological severity, and when the screening of bacterial infections was negative (2). Treatment was discontinued when patients were nonresponders to steroids at day 7 upon a Lille score ≥ 0,45 (15). In selected patients, liver transplantation was indicated as previously described (17).
Data collection The following characteristics were collected at baseline: age, gender, number of previous AAH episodes, presence of hepatic encephalopathy and cirrhosis. Biological markers were collected at the time of admission but also at the time of hospitalization in the primary care center: prothrombin rate, international normalized ratio (INR), bilirubin and, creatinine levels. MDF and MELD scores were calculated. The delay between sample collection and AAH diagnosis was measured and sepsis occurrence was reported. When indicated, the use and response to steroids (Lille score) were documented. Adverse events and outcome were reported (survival and liver transplantation). Serum samples were collected during
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hospitalization for routine diagnosis and were stored at -80°C. Investigations were conducted upon agreement of the local ethical committee.
Virological analysis: HEV markers were searched retrospectively on stored sera. HEV serology (IgM and IgG) was performed by using the Wantai assays (Beijing, China). After total nucleic acids extraction (NucliSens® easyMAG™, Biomérieux, Marcy l’étoile, France), HEV RNA was detected by using the hepatitisE@ceeramTools assay (Ceeram, La Chapelle sur Erdre, France). HEV genotyping was determined from the phylogenetic analysis of open reading frame (ORF)-2 region, as described (18) and routinely performed at the French National Reference Centre for HAV and HEV. Acute HEV infection was defined as positive IgM and/or positive HEV RNA. In case of positivity, risks factors were investigated. For each patient, serological markers of ongoing viral hepatitis A, B, and C and Epstein-Barr virus and cytomegalovirus viremias were routinely performed.
Histological analysis: All liver biopsies were reviewed retrospectively by a single pathologist. AAH was histologically defined by the presence of apoptotic hepatocytes accompanied by a lobular inflammation composed of polynuclear neutrophils. Other features including macrovacuolar steatosis, hepatocyte ballooning and Mallory bodies were recorded as well as the assessment of an underlying chronic hepatitis related to alcohol consumption such as fibrosis (from portal fibrosis to cirrhosis) and perisinusoidal fibrosis. The severity of acute
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lesions was graded as mild, moderate or severe. The presence of cholestasis in cholangiolar ductules was suggestive of sepsis.
Data analysis: Patients’ characteristics were described using percentages for categorical variables and mean ± standard-deviation for continuous variables. The Mann Whitney U test and Fisher's exact test were performed under Statistica version 6 software (Stat Soft France, 2004), when appropriate.
RESULTS: General characteristics and outcomes Among the 156 patients admitted in our center for severe AAH from 2007 to 2013, 72 were excluded from the final analysis: 2 were transplant patients, 39 had no available sera, 25 had no available liver biopsy, and in 6 patients liver biopsy was not consistent with AAH features. Eighty-four patients were finally included. All, but one, had been previously hospitalized in primary care centers and were secondarily referred due to clinical severity. Patients’ characteristics are shown on Table 1. Mean age was 50.8±9.6 years, 65.5% were male, 91.7% were cirrhotic and 33.3% presented with encephalopathy. Mean MELD score and MDF were 32.4±11.4 and 73.3±37, respectively. Bacterial infections were reported in 29 (34.5%) cases. Liver biopsies showed minimal or mild acute hepatitis in 25 patients (29.8%), moderate in 23 (27.4%) and severe in 32(38,1%). Suggestive histological features of sepsis were observed in 26 (32.5%) patients. Four biopsies were performed in the primary care center and could not be reviewed and graded by the expert pathologist in our center. These This article is protected by copyright. All rights reserved.
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four patients had a severe presentation with a mean (±SD) MDF of 84±20 and all received steroids. Steroids were given to 61 (72.6%) patients, including 12(48%), 18(78%) and 27(84%) with mild, moderate and severe histological features of AAH, respectively. Thirtyfive (57.4%) patients were non-responders with a mean Lille score of 0.78±0.21. During hospitalization, 24 (28.6%) of the patients died and 11 (13.1%) were transplanted.
Virological analyses As shown in Table 1, 60 patients (71.4%) had a negative HEV serology (IgM-/IgG-), 21 (25%) had markers of past infection (IgM-/IgG+), and 3 (3.6%) had markers of acute infection (IgM+/IgG+). HEV RNA was undetectable in patients from the first two groups and was detectable in 2/3 patients with positive IgM. A 3c HEV genotype could be obtained in one case. Mean delay between sample collection and HEV diagnosis was 4.7±32.8 days and was not different between the obtained serological profiles (10±19.1, -10.5±55.4 and 3.70±5.5 days for IgM-/IgG-, IgM-/IgG+ and IgM+/IgG+ patients, respectively). Routine investigations for other active viral infections were negative.
Acute HEV infection in AAH patients Table 2 specifies the clinical features of the 3 patients with acute HEV infection markers. HEV infection was diagnosed retrospectively in patient 1, since he was admitted in 2007 when HEV serology was not routinely performed. On the liver biopsy showing cirrhosis, acute hepatitis was mild. No sign suggestive of sepsis was observed. Despite a non-response to steroids, the outcome was favorable. In patient 2, HEV infection was diagnosed after 7 This article is protected by copyright. All rights reserved.
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days of steroid treatment, for which he was non-responder. On the liver biopsy showing extensive fibrosis, acute hepatitis was severe. No sign suggestive of sepsis was observed. Despite a high MELD score at baseline and the absence of response to steroids, the outcome was slowly favorable and the patient could discharge. He is currently still abstinent and perfectly compensated. In patient 3, the diagnosis of acute HEV infection was made soon after admission, and liver biopsy showed cirrhosis and acute moderate hepatitis. In addition to AAH features the liver biopsy showed a lymphoplasmacytic infiltrate indicating a possible association with a viral infection (Figure 1). No sign suggestive of sepsis was observed. Despite a MDF > 100, steroids were not introduced because of a septic shock, leading to death 8 days after patient’s admission in the intensive care unit. For these patients, no factors of HEV transmission could be identified (no travel history, no transfusion), arguing in favor of a foodborne transmission. Both HEV-positive treated patients were non-responders to steroids with very high Lille scores (0.93 and 0.95 for patients 1 and 2, respectively).
Comparison between HEV-infected and uninfected patients. No significant differences in terms of clinical presentation, demographic characteristics or outcome were observed between patients with or without acute HEV. In particular, similar mean MELD score and MDF were observed: at admission in the primary care center, mean MELD was 39.3±14.6 vs. 28.6±8.3 (p=0.09) and Maddrey score was 78.3±19 vs. 64.6±33.9 (p=0.16), respectively in HEV IgM+ vs. IgM- patients. Nevertheless, although numbers are low, patients with acute HEV presented higher bilirubin: 607±159.5 vs. 294±179.5 µmol/L (p=0.009) and higher creatinine levels: 154±21 vs. 111±127 µmol/L (p=0.028) at admission in the primary care center and the delay before referral was shorter: 6±2 vs. 19.4±13.8 days This article is protected by copyright. All rights reserved.
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(p=0.04). Regarding outcome, no significant differences in terms of steroid response rate and death were observed.
DISCUSSION: The present study is the first to evaluate HEV burden in the specific setting of ACLF due to AAH. Markers of acute HEV infection were present in 3.6% of patients admitted for histologically proven AAH in our center.
Mortality due to liver disease and the consumption of alcohol and pork were previously described as being linked in Southern England (8), and autochthonous HEV infection is presumably mostly transmitted by food intake (including pork and delicatessen). Patients with alcohol consumption may also be those at risk of HEV infection and HEV could represent a precipitating factor of acute liver failure. Indeed, in highly endemic countries, it has been shown that HEV infection was an independent factor for rapid liver decompensation and high rate mortality (7). Conversely, a previous study form the French Midi-Pyrénées region reported seven cases of severe HEV infections of which 5 occurred in patients with active alcohol abuse and chronic liver disease (9). We then hypothesized that HEV infection could account for the severity liver disease decompensation in the specific setting of AAH, and the observed corticoresistance. In the present study, we indeed observed some cases of superimposed HEV infections in patients with AAH, and, though pronounced renal impairment may affect bilirubin levels, HEV infection can also have contributed to the higher bilirubin levels observed in these patients. However, the number This article is protected by copyright. All rights reserved.
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of HEV infected patients is weak and limits any conclusions regarding patients’ prognosis. In addition, the clinical severity was globally much higher than in previously published series, which makes more difficult highlighting factors that would contribute to an increased severity (15, 19, 20).
The seroprevalence of hepatitis E in France is one of the most elevated in Europe with England and Denmark (21), although a high disparity exists in the country. Seroprevalence rates reported in 2007 were 3% in Ile-de-France region and 16.6% in the South West France (22) and a recent study estimated the global seroprevalence as 5% (23). However, 3 to 4fold higher rates were described by using more sensitive methods (24), reaching 52.5% in South-West France, and increasing with age, rates of 70% in people over 58 years old (21). The reasons for these extremely high rates are not clearly understood, but may partly be related to dietary habits, which include eating pork and also suggests that HEV infection is mostly asymptomatic or poorly symptomatic in immunocompetent subjects. Data evaluating seroprevalence in the North France using sensitive techniques are lacking, but it can be estimated that in Ile de France region, overall HEV seroprevalence would reach 1520%, with sensitive assays such as the one used in this study. A seroprevalence rate of 28.6% in patients with a mean age of 50 years is consistent with this estimation. The patients of this study, who experienced acute HEV infection during the present AAH episode or who present markers of past infection, have probably been infected by food intake, and notably pork. Indeed, it has been shown that HEV genotype 3 strains circulate in a significant proportion of pig farms in Europe (25-27).
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HEV diagnosis is challenging because it is commonly performed after exclusion of other causes of hepatitis, including viral infections (hepatitis A, B, C, CMV, EBV) (13) though autochthonous HEV infections are increasingly reported in developed countries (28). None of HEV IgM-positive patients reported a travel abroad. Another point highlighted by this study is the need for histological assessment of clinical severe AAH for appropriate management of these patients. Indeed, histology allowed evidencing both alcohol- and virus-related liver injury in one case. The search for additional causes of ACLF in patient presenting with a clinical syndrome resembling AAH, including HEV, is of major importance. Indeed, steroids are a treatment option for severe AAH and it is well known that immunosuppressed patients may develop chronic HEV infection (12,29). Therefore, HEV diagnosis could potentially change the management of AAH. However, it has to be noted that no persistent HEV infection occurred in the 2 patients who received steroids in the present study. These two patients were both resistant to steroids. Although no conclusion can be drawn from so small numbers, this finding points to the need for larger studies to assess the potential impact of concomitant HEV infection on steroids response.
In conclusion, a substantial proportion of patients with ACLF due to AAH had also markers of acute HEV infection. In patients presenting with particularly severe AAH, concomitant HEV infection was not associated with significant worse outcome. Although our study is retrospective with a certain number of limitations such as the small number of events, it allows us to obtain important information that should be known to physicians in our field. Heavy drinkers or patients with chronic liver diseases should be aware of the risk of hepatitis E infection and the recommendation to avoid raw or undercooked pork This article is protected by copyright. All rights reserved.
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consumption should be extended to these patients. In addition, an effort should be made for the routine screening of hepatitis E in this context. Finally, even if we can not recommend avoiding steroids, early diagnosis of hepatitis E associated with AAH could lead to consider HEV treatment in the absence of spontaneous clearance. Therefore, larger studies are required to evaluate further HEV burden in AAH and impact on steroid response, to assess the potential benefit of HEV treatment by ribavirin in this setting, and the risk of persistent HEV infection in these immunosuppressed cirrhotic patients.
Acknowledgments We thank Claire Mony for her technical assistance. We thank all the patients and families and all the team of the Hepato-Biliary Center of Paul Brousse Hospital.
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seroprevalence in blood donors, the elderly and patients with chronic liver disease. European journal of gastroenterology & hepatology 2008; 20(8): 784-90. 9. PERON J M, BUREAU C, POIRSON H, et al. Fulminant liver failure from acute autochthonous hepatitis E in France: description of seven patients with acute hepatitis E and encephalopathy. Journal of viral hepatitis 2007; 14(5): 298-303. 10. DALTON H R. Hepatitis: hepatitis E and decompensated chronic liver disease. Nat Rev Gastroenterol Hepatol 2012; 9(8): 430-2. 11. DALTON H R, BENDALL R P, PRITCHARD C, HENLEY W, MELZER D. National mortality rates from chronic liver disease and consumption of alcohol and pig meat. Epidemiol Infect 2010; 138(2): 174-82. 12. KAMAR N, SELVES J, MANSUY J M, et al. Hepatitis E virus and chronic hepatitis in organtransplant recipients. N Engl J Med 2008; 358(8): 811-7. 13. DAVERN T J, CHALASANI N, FONTANA R J, et al. Acute hepatitis E infection accounts for some cases of suspected drug-induced liver injury. Gastroenterology 2011; 141(5): 1665-72 e1-9. 14. HAIM-BOUKOBZA S, FEREY M P, VETILLARD A L, et al. Transfusion-transmitted hepatitis E in a misleading context of autoimmunity and drug-induced toxicity. Journal of hepatology 2012. 15. LOUVET A, NAVEAU S, ABDELNOUR M, et al. The Lille model: a new tool for therapeutic strategy in patients with severe alcoholic hepatitis treated with steroids. Hepatology (Baltimore, Md 2007; 45(6): 1348-54. 16. LIVER E A F T S O T. EASL clinical practical guidelines: management of alcoholic liver disease. Journal of hepatology 2012; 57(2): 399-420. 17. MATHURIN P, MORENO C, SAMUEL D, et al. Early liver transplantation for severe alcoholic hepatitis. N Engl J Med 2011; 365(19): 1790-800. 18. COOPER K, HUANG F F, BATISTA L, et al. Identification of genotype 3 hepatitis E virus (HEV) in serum and fecal samples from pigs in Thailand and Mexico, where genotype 1 and 2 HEV strains are prevalent in the respective human populations. J Clin Microbiol 2005; 43(4): 1684-8. 19. MATHURIN P, LOUVET A, DUHAMEL A, et al. Prednisolone with vs without pentoxifylline and survival of patients with severe alcoholic hepatitis: a randomized clinical trial. JAMA 2013; 310(10): 1033-41. 20. MATHURIN P, O'GRADY J, CARITHERS R L, et al. Corticosteroids improve short-term survival in patients with severe alcoholic hepatitis: meta-analysis of individual patient data. Gut 2011; 60(2): 255-60. 21. MANSUY J M, BENDALL R, LEGRAND-ABRAVANEL F, et al. Hepatitis E virus antibodies in blood donors, France. Emerg Infect Dis 2011; 17(12): 2309-12. 22. MANSUY J M, LEGRAND-ABRAVANEL F, CALOT J P, et al. High prevalence of anti-hepatitis E virus antibodies in blood donors from South West France. J Med Virol 2008; 80(2): 289-93. 23. LEPOUTRE AGNÈS A D, FONTENEAU LAURE, HALFTERMEYER-ZHOU FANGQIN, BAUDON CLAIRE, DORLÉANS FRÉDÉRIQUE, LE STRAT YANN, LÉVY-BRUHL DANIEL. Séroprévalence des maladies à prévention vaccinale et de cinq autres maladies infectieuses en France. Résultat de deux enquêtes nationales 2008-2010. Bulletin épidémiologique hebdomadaire 2013; 41-42(10 décembre 2013): 526-34. 24. KAMAR N, DALTON H R, ABRAVANEL F, IZOPET J. Hepatitis e virus infection. Clin Microbiol Rev 2014; 27(1): 116-38. 25. BERTO A, BACKER J A, MESQUITA J R, et al. Prevalence and transmission of hepatitis E virus in domestic swine populations in different European countries. BMC Res Notes 2012; 5: 190. 26. BERTO A, GRIERSON S, HAKZE-VAN DER HONING R, et al. Hepatitis E virus in pork liver sausage, France. Emerg Infect Dis 2013; 19(2): 264-6. 27. BERTO A, MARTELLI F, GRIERSON S, BANKS M. Hepatitis E virus in pork food chain, United Kingdom, 2009-2010. Emerg Infect Dis 2012; 18(8): 1358-60. 28. DALTON H R, STABLEFORTH W, HAZELDINE S, et al. Autochthonous hepatitis E in Southwest England: a comparison with hepatitis A. Eur J Clin Microbiol Infect Dis 2008; 27(7): 579-85.
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29. KAMAR N, ROSTAING L, ABRAVANEL F, et al. Ribavirin therapy inhibits viral replication on patients with chronic hepatitis e virus infection. Gastroenterology 2010; 139(5): 1612-8.
Legends to Table and figures: Table 1: Clinical characteristics of the patients according to the results of HEV serology
Table 2: Clinical characteristics of the patients presenting with markers of acute HEV infection
Figure 1: Figure 1A: Liver biopsy stained with picrosirius (case 3). Cirrhosis was accompanied with perisinusoidal fibrosis. Note that the sinusoids are dilated and fulfilled with numerous cells. Figure 1B: Liver biopsy stained with hematein eosin safran (case 3). Alcohol-related features include steatosis, hepatocyte ballooning, Malory bodies (red arrow), foci of neutrophils (blue arrow). Surprisingly, intrasinusoidal infiltrate is rich and composed of lymphocytes and plasma cells (green arrows).
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Received Date : 25-Mar-2014 Revised Date : 26-May-2014 Accepted Date : 30-May-2014 Article type
: Original Articles
Hepatitis E infection in patients with severe acute alcoholic hepatitis HAIM-BOUKOBZA Stéphanie (1,2,3,6), COILLY Audrey (2,3,4,6), SEBAGH Mylène (3,5,6), BOUAMOUD Mouna (4), ANTONINI Teresa (3,4,6), ROCHE Bruno (3,4,6), YORDANOVA Olga (1), SAVARY Janine (1), SALIBA Faouzi (2,3,4,6), DUCLOS-VALLEE Jean-Charles (2,3,4,6), SAMUEL Didier (2,3,4,6), ICHAI Philippe (2,3,4,6), ROQUE-AFONSO Anne-Marie (1,2,3,6)
1
AP-HP, Hôpital Paul Brousse, Virologie, CNR des hépatites A et E, Villejuif, 94804 France
2
Univ Paris-Sud, UMR-S 785, Le Kremlin Bicêtre, 94275 France
3
INSERM U785, Villejuif, 94804 France
4
AP-HP, Hôpital Paul Brousse, Centre Hépato-Biliaire, Villejuif, 94804 France
5
AP-HP, Hôpital Paul Brousse, Service d’anatomie pathologie, Villejuif 94804
6
Hepatinov, Villejuif F94800, France
Corresponding author: Mailing address : Pr ROQUE-AFONSO Anne-Marie 12-14 av Paul Vaillant Couturier 94804 Villejuif Cedex France This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/liv.12610 This article is protected by copyright. All rights reserved.
Accepted Article
[email protected] Phone : + 33 1 45 59 37 21 Fax : + 33 1 45 59 37 24
ABSTRACT: BACKGROUND: Hepatitis E virus (HEV) infection is a known cause of acute on chronic liver failure in developing countries, but its implication in Western countries remains unknown.
AIMS: To assess HEV burden in the setting of severe acute alcoholic hepatitis (AAH).
METHODS: Patients admitted for severe AAH from 2007 to 2013, with available sera and histologically proven AAH, were included and managed according to current European guidelines. At admission, clinical and biological characteristics were collected; HEV serology and RNA detection were retrospectively performed.
RESULTS: Eighty-four patients were included. Mean age was 50.8±9.6 years, 65.5% were male, 91.7% were cirrhotic and 33.3% presented with encephalopathy. Mean MELD and Maddrey scores were respectively 32.4±11.4 and 73.3±37. Liver biopsy showed mild, moderate and severe hepatitis in 25 (29.8%), 23 (27.4%) and 32 (38.1%) patients, respectively. Steroids were given to 61 patients (72.6%) of whom 35 (57.4%) presented corticoresistance (mean Lille score: 0.78±0.21). During hospitalization, 24 patients (28.6%) died and 11 (13.1%) were transplanted. Three patients (3.6%) presented markers of acute HEV infection and 21 (25%), markers of past HEV infection. Patient with acute infection were men, cirrhotic, and 2/3 presented with encephalopathy. Steroids were given to 2 patients without any response. The third patient died. None were transplanted.
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CONCLUSIONS: A substantial proportion of patients with severe AAH had markers of acute HEV infection, with similar clinical presentation and outcomes. Larger studies are needed to evaluate HEV impact on AAH management, resistance to steroids, and outcome.
Key words: hepatitis E, acute alcoholic hepatitis, Lille score, corticoresistance, Maddrey score, MELD score, cirrhosis.
ARTICLE Alcoholic liver disease is the most prevalent cause of advanced liver disease in Europe (1). Hepatic manifestations include alcoholic fatty liver disease, acute alcoholic hepatitis (AAH) and cirrhosis. The majority of patients with AAH have a history of heavy alcohol use and concomitant cirrhosis is present in about 50%-60% cases (2). The clinical syndrome is characterized by a recent onset of deep jaundice, which can lead to an end stage liver failure. Symptoms are usually non-specific including fatigue, fever, ascites and encephalopathy. The diagnosis is based on histological features (i.e. steatosis, hepatocyte injury and neutrophilic infiltration). The overall 30-day mortality rate is approximately 15%. However, in patients with severe liver disease defined by a Maddrey’s discriminant (MDF) function ≥32, the rate can reach 50 % (3, 4). Short-term prognosis of patients with a severe AAH has been improved with steroid use (5).
Hepatitis E virus (HEV) infection is a cause of acute liver injury. In low-income countries with poor sanitation and hygiene, HEV is a common cause of acute hepatitis transmitted through contaminated water. In these countries, infection is due to genotypes 1 and 2 that exclusively infect humans. In industrialized countries, HEV is responsible for sporadic cases This article is protected by copyright. All rights reserved.
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due to genotypes 3 and 4 that also infect other animals, mainly pigs, and the transmission appears to be zoonotic or foodborne (6). HEV infection has been shown to account significantly for acute-on-chronic liver failure (ACLF), predominantly in patients from the Indian subcontinent, and it was shown that cirrhotic patients who experienced a superimposed HEV infection had a higher risk of rapid decompensation and death (7). The occurrence and outcome of HEV infection in patients presenting with ACLF is still poorly described in Western countries, especially in the population of patients with AAH (8, 9). Nevertheless, several facts argue for a systematic screening of HEV infection in patients with decompensated alcoholic liver disease in Western countries: 1-as in endemic countries, HEV infection could enhance acute or sub-acute liver failure in patients with pre-existing liver disease (9); 2-mortality due to chronic liver disease has been linked to the consumption of pork and alcohol in developed countries (10, 11); 3-there is a potential risk of developing a chronic HEV infection considering the immunosuppression status of AAH patients due to steroids (12). Hepatitis E is still not commonly diagnosed in the first line of ACLF in most centers, and is often an exclusion diagnosis (13, 14). The aim of this retrospective study was to assess the frequency of HEV infection and its prognosis in patients admitted for severe AAH in a single center.
PATIENTS AND METHODS: Patients’ selection All patients admitted in the hepato-biliary center of the Paul Brousse hospital from 2007 to 2013 for severe AAH were considered for the present study. Severity was defined as a MDF
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≥ 32 and/or a MELD score ≥ 20. Patients previously transplanted were excluded and only patients with histologically proven AAH and available serum were included.
Clinical management of AAH AAH management was based on current guidelines (15, 16). Psychotherapy to maintain abstinence was early initiated. A transjugular liver biopsy was systematically considered when MDF was ≥ 32. Patients with severe clinical presentation (MDF ≥ 32) were considered for treatment with prednisolone 40mg/day during 28 days in case of histologically proven AAH, whatever the degree of histological severity, and when the screening of bacterial infections was negative (2). Treatment was discontinued when patients were nonresponders to steroids at day 7 upon a Lille score ≥ 0,45 (15). In selected patients, liver transplantation was indicated as previously described (17).
Data collection The following characteristics were collected at baseline: age, gender, number of previous AAH episodes, presence of hepatic encephalopathy and cirrhosis. Biological markers were collected at the time of admission but also at the time of hospitalization in the primary care center: prothrombin rate, international normalized ratio (INR), bilirubin and, creatinine levels. MDF and MELD scores were calculated. The delay between sample collection and AAH diagnosis was measured and sepsis occurrence was reported. When indicated, the use and response to steroids (Lille score) were documented. Adverse events and outcome were reported (survival and liver transplantation). Serum samples were collected during
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hospitalization for routine diagnosis and were stored at -80°C. Investigations were conducted upon agreement of the local ethical committee.
Virological analysis: HEV markers were searched retrospectively on stored sera. HEV serology (IgM and IgG) was performed by using the Wantai assays (Beijing, China). After total nucleic acids extraction (NucliSens® easyMAG™, Biomérieux, Marcy l’étoile, France), HEV RNA was detected by using the hepatitisE@ceeramTools assay (Ceeram, La Chapelle sur Erdre, France). HEV genotyping was determined from the phylogenetic analysis of open reading frame (ORF)-2 region, as described (18) and routinely performed at the French National Reference Centre for HAV and HEV. Acute HEV infection was defined as positive IgM and/or positive HEV RNA. In case of positivity, risks factors were investigated. For each patient, serological markers of ongoing viral hepatitis A, B, and C and Epstein-Barr virus and cytomegalovirus viremias were routinely performed.
Histological analysis: All liver biopsies were reviewed retrospectively by a single pathologist. AAH was histologically defined by the presence of apoptotic hepatocytes accompanied by a lobular inflammation composed of polynuclear neutrophils. Other features including macrovacuolar steatosis, hepatocyte ballooning and Mallory bodies were recorded as well as the assessment of an underlying chronic hepatitis related to alcohol consumption such as fibrosis (from portal fibrosis to cirrhosis) and perisinusoidal fibrosis. The severity of acute
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lesions was graded as mild, moderate or severe. The presence of cholestasis in cholangiolar ductules was suggestive of sepsis.
Data analysis: Patients’ characteristics were described using percentages for categorical variables and mean ± standard-deviation for continuous variables. The Mann Whitney U test and Fisher's exact test were performed under Statistica version 6 software (Stat Soft France, 2004), when appropriate.
RESULTS: General characteristics and outcomes Among the 156 patients admitted in our center for severe AAH from 2007 to 2013, 72 were excluded from the final analysis: 2 were transplant patients, 39 had no available sera, 25 had no available liver biopsy, and in 6 patients liver biopsy was not consistent with AAH features. Eighty-four patients were finally included. All, but one, had been previously hospitalized in primary care centers and were secondarily referred due to clinical severity. Patients’ characteristics are shown on Table 1. Mean age was 50.8±9.6 years, 65.5% were male, 91.7% were cirrhotic and 33.3% presented with encephalopathy. Mean MELD score and MDF were 32.4±11.4 and 73.3±37, respectively. Bacterial infections were reported in 29 (34.5%) cases. Liver biopsies showed minimal or mild acute hepatitis in 25 patients (29.8%), moderate in 23 (27.4%) and severe in 32(38,1%). Suggestive histological features of sepsis were observed in 26 (32.5%) patients. Four biopsies were performed in the primary care center and could not be reviewed and graded by the expert pathologist in our center. These This article is protected by copyright. All rights reserved.
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four patients had a severe presentation with a mean (±SD) MDF of 84±20 and all received steroids. Steroids were given to 61 (72.6%) patients, including 12(48%), 18(78%) and 27(84%) with mild, moderate and severe histological features of AAH, respectively. Thirtyfive (57.4%) patients were non-responders with a mean Lille score of 0.78±0.21. During hospitalization, 24 (28.6%) of the patients died and 11 (13.1%) were transplanted.
Virological analyses As shown in Table 1, 60 patients (71.4%) had a negative HEV serology (IgM-/IgG-), 21 (25%) had markers of past infection (IgM-/IgG+), and 3 (3.6%) had markers of acute infection (IgM+/IgG+). HEV RNA was undetectable in patients from the first two groups and was detectable in 2/3 patients with positive IgM. A 3c HEV genotype could be obtained in one case. Mean delay between sample collection and HEV diagnosis was 4.7±32.8 days and was not different between the obtained serological profiles (10±19.1, -10.5±55.4 and 3.70±5.5 days for IgM-/IgG-, IgM-/IgG+ and IgM+/IgG+ patients, respectively). Routine investigations for other active viral infections were negative.
Acute HEV infection in AAH patients Table 2 specifies the clinical features of the 3 patients with acute HEV infection markers. HEV infection was diagnosed retrospectively in patient 1, since he was admitted in 2007 when HEV serology was not routinely performed. On the liver biopsy showing cirrhosis, acute hepatitis was mild. No sign suggestive of sepsis was observed. Despite a non-response to steroids, the outcome was favorable. In patient 2, HEV infection was diagnosed after 7 This article is protected by copyright. All rights reserved.
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days of steroid treatment, for which he was non-responder. On the liver biopsy showing extensive fibrosis, acute hepatitis was severe. No sign suggestive of sepsis was observed. Despite a high MELD score at baseline and the absence of response to steroids, the outcome was slowly favorable and the patient could discharge. He is currently still abstinent and perfectly compensated. In patient 3, the diagnosis of acute HEV infection was made soon after admission, and liver biopsy showed cirrhosis and acute moderate hepatitis. In addition to AAH features the liver biopsy showed a lymphoplasmacytic infiltrate indicating a possible association with a viral infection (Figure 1). No sign suggestive of sepsis was observed. Despite a MDF > 100, steroids were not introduced because of a septic shock, leading to death 8 days after patient’s admission in the intensive care unit. For these patients, no factors of HEV transmission could be identified (no travel history, no transfusion), arguing in favor of a foodborne transmission. Both HEV-positive treated patients were non-responders to steroids with very high Lille scores (0.93 and 0.95 for patients 1 and 2, respectively).
Comparison between HEV-infected and uninfected patients. No significant differences in terms of clinical presentation, demographic characteristics or outcome were observed between patients with or without acute HEV. In particular, similar mean MELD score and MDF were observed: at admission in the primary care center, mean MELD was 39.3±14.6 vs. 28.6±8.3 (p=0.09) and Maddrey score was 78.3±19 vs. 64.6±33.9 (p=0.16), respectively in HEV IgM+ vs. IgM- patients. Nevertheless, although numbers are low, patients with acute HEV presented higher bilirubin: 607±159.5 vs. 294±179.5 µmol/L (p=0.009) and higher creatinine levels: 154±21 vs. 111±127 µmol/L (p=0.028) at admission in the primary care center and the delay before referral was shorter: 6±2 vs. 19.4±13.8 days This article is protected by copyright. All rights reserved.
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(p=0.04). Regarding outcome, no significant differences in terms of steroid response rate and death were observed.
DISCUSSION: The present study is the first to evaluate HEV burden in the specific setting of ACLF due to AAH. Markers of acute HEV infection were present in 3.6% of patients admitted for histologically proven AAH in our center.
Mortality due to liver disease and the consumption of alcohol and pork were previously described as being linked in Southern England (8), and autochthonous HEV infection is presumably mostly transmitted by food intake (including pork and delicatessen). Patients with alcohol consumption may also be those at risk of HEV infection and HEV could represent a precipitating factor of acute liver failure. Indeed, in highly endemic countries, it has been shown that HEV infection was an independent factor for rapid liver decompensation and high rate mortality (7). Conversely, a previous study form the French Midi-Pyrénées region reported seven cases of severe HEV infections of which 5 occurred in patients with active alcohol abuse and chronic liver disease (9). We then hypothesized that HEV infection could account for the severity liver disease decompensation in the specific setting of AAH, and the observed corticoresistance. In the present study, we indeed observed some cases of superimposed HEV infections in patients with AAH, and, though pronounced renal impairment may affect bilirubin levels, HEV infection can also have contributed to the higher bilirubin levels observed in these patients. However, the number This article is protected by copyright. All rights reserved.
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of HEV infected patients is weak and limits any conclusions regarding patients’ prognosis. In addition, the clinical severity was globally much higher than in previously published series, which makes more difficult highlighting factors that would contribute to an increased severity (15, 19, 20).
The seroprevalence of hepatitis E in France is one of the most elevated in Europe with England and Denmark (21), although a high disparity exists in the country. Seroprevalence rates reported in 2007 were 3% in Ile-de-France region and 16.6% in the South West France (22) and a recent study estimated the global seroprevalence as 5% (23). However, 3 to 4fold higher rates were described by using more sensitive methods (24), reaching 52.5% in South-West France, and increasing with age, rates of 70% in people over 58 years old (21). The reasons for these extremely high rates are not clearly understood, but may partly be related to dietary habits, which include eating pork and also suggests that HEV infection is mostly asymptomatic or poorly symptomatic in immunocompetent subjects. Data evaluating seroprevalence in the North France using sensitive techniques are lacking, but it can be estimated that in Ile de France region, overall HEV seroprevalence would reach 1520%, with sensitive assays such as the one used in this study. A seroprevalence rate of 28.6% in patients with a mean age of 50 years is consistent with this estimation. The patients of this study, who experienced acute HEV infection during the present AAH episode or who present markers of past infection, have probably been infected by food intake, and notably pork. Indeed, it has been shown that HEV genotype 3 strains circulate in a significant proportion of pig farms in Europe (25-27).
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HEV diagnosis is challenging because it is commonly performed after exclusion of other causes of hepatitis, including viral infections (hepatitis A, B, C, CMV, EBV) (13) though autochthonous HEV infections are increasingly reported in developed countries (28). None of HEV IgM-positive patients reported a travel abroad. Another point highlighted by this study is the need for histological assessment of clinical severe AAH for appropriate management of these patients. Indeed, histology allowed evidencing both alcohol- and virus-related liver injury in one case. The search for additional causes of ACLF in patient presenting with a clinical syndrome resembling AAH, including HEV, is of major importance. Indeed, steroids are a treatment option for severe AAH and it is well known that immunosuppressed patients may develop chronic HEV infection (12,29). Therefore, HEV diagnosis could potentially change the management of AAH. However, it has to be noted that no persistent HEV infection occurred in the 2 patients who received steroids in the present study. These two patients were both resistant to steroids. Although no conclusion can be drawn from so small numbers, this finding points to the need for larger studies to assess the potential impact of concomitant HEV infection on steroids response.
In conclusion, a substantial proportion of patients with ACLF due to AAH had also markers of acute HEV infection. In patients presenting with particularly severe AAH, concomitant HEV infection was not associated with significant worse outcome. Although our study is retrospective with a certain number of limitations such as the small number of events, it allows us to obtain important information that should be known to physicians in our field. Heavy drinkers or patients with chronic liver diseases should be aware of the risk of hepatitis E infection and the recommendation to avoid raw or undercooked pork This article is protected by copyright. All rights reserved.
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consumption should be extended to these patients. In addition, an effort should be made for the routine screening of hepatitis E in this context. Finally, even if we can not recommend avoiding steroids, early diagnosis of hepatitis E associated with AAH could lead to consider HEV treatment in the absence of spontaneous clearance. Therefore, larger studies are required to evaluate further HEV burden in AAH and impact on steroid response, to assess the potential benefit of HEV treatment by ribavirin in this setting, and the risk of persistent HEV infection in these immunosuppressed cirrhotic patients.
Acknowledgments We thank Claire Mony for her technical assistance. We thank all the patients and families and all the team of the Hepato-Biliary Center of Paul Brousse Hospital.
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Legends to Table and figures: Table 1: Clinical characteristics of the patients according to the results of HEV serology
Table 2: Clinical characteristics of the patients presenting with markers of acute HEV infection
Figure 1: Figure 1A: Liver biopsy stained with picrosirius (case 3). Cirrhosis was accompanied with perisinusoidal fibrosis. Note that the sinusoids are dilated and fulfilled with numerous cells. Figure 1B: Liver biopsy stained with hematein eosin safran (case 3). Alcohol-related features include steatosis, hepatocyte ballooning, Malory bodies (red arrow), foci of neutrophils (blue arrow). Surprisingly, intrasinusoidal infiltrate is rich and composed of lymphocytes and plasma cells (green arrows).
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